Structural Insight into DNA-Dependent Activation of Human Metalloprotease Spartan

Faxiang Li,Joanna E Raczynska,Zhe Chen,Hongtao Yu

doi:10.1016/j.celrep.2019.02.082

Abstract

The DNA-dependent metalloprotease Spartan (SPRTN) cleaves DNA-protein crosslinks (DPCs) and protects cells from DPC-induced genome instability. Germline mutations of SPRTN are linked to human Ruijs-Aalfs syndrome (RJALS) characterized by progeria and early-onset hepatocellular carcinoma. The mechanism of DNA-mediated activation of SPRTN is not understood. Here, we report the crystal structure of the human SPRTN SprT domain bound to single-stranded DNA (ssDNA). Our structure reveals a Zn2+-binding sub-domain (ZBD) in SprT that shields its active site located in the metalloprotease sub-domain (MPD). The narrow catalytic groove between MPD and ZBD only permits cleavage of flexible substrates. The ZBD contains an ssDNA-binding site, with a DNA-base-binding pocket formed by aromatic residues. Mutations of ssDNA-binding residues diminish the protease activity of SPRTN. We propose that the ZBD contributes to the ssDNA specificity of SPRTN, restricts the access of globular substrates, and positions DPCs, which may need to be partially unfolded, for optimal cleavage.

Highlights

Genomic DNA is constantly subjected to many types of damage, including single- or double-stranded breaks, bulky adducts, intra- and interstrand crosslinks, and DNA-protein crosslinks (DPCs), which are caused by various endogenous and exogenous factors (Hoeijmakers, 2009; Stingele et al, 2017; Vaz et al, 2017)
We confirm and extend previous findings that SPRTN is preferentially activated by single-stranded DNA (ssDNA)
Our findings provide a possible explanation for the ssDNA specificity of SPRTN and suggest that SPRTN preferentially acts on DPCs at DNA replication forks, which contain ssDNA

Summary

Introduction

Genomic DNA is constantly subjected to many types of damage, including single- or double-stranded breaks, bulky adducts, intra- and interstrand crosslinks, and DNA-protein crosslinks (DPCs), which are caused by various endogenous and exogenous factors (Hoeijmakers, 2009; Stingele et al, 2017; Vaz et al, 2017). Proteomic analyses of DPCs reveal that numerous DNA-binding proteins, including transcription factors, DNA repair and replication proteins, histones, and non-DNAbinding proteins, contribute to DPC formation (Lai et al, 2016; Loeber et al, 2009). Non-enzymatic DPCs are formed by the crosslinking of proteins to DNA by UV light, ionizing radiation, or reactive aldehydes. Enzymatic DPCs are formed by the stable trapping of transient covalent protein-DNA intermediates during the reaction cycles of DNA enzymes. The inhibition of topoisomerases 1 or 2 (Top1/2) by campothecin or etoposide produces DPCs between the DNA backbone and the catalytic tyrosine (Pommier, 2013)

Methods

Results

Discussion

Conclusion